To understand the sibship of Notholirion and the genetic differentiation of N.bulbuliferum,the genetic diversity of 255 individuals from 24 populations was analyzed by using seven intersimple sequence repeat (ISSR) markers.(1)The results of the UPGMA analysis exhibited significant genetic differentiation between N.bulbuliferum,N.campanulatum,and N.macrophyllum,which indicated that they are separate species.(2)N.bulbuliferum×N.campanulatum clustered together with N.campanulatum,which may be a result from the matrilineal inheritance and strong asexual reproduction capacity;its low gene flow (N_m=0.216 0) implied that N.bulbuliferum×N.campanulatum may be undergoing differentiation.(3)The Mantel test showed a substantial significant correlation between genetic and geographic distances in N.bulbuliferum (r=0.410,P=0.01),and clustered four groups.(4)The analysis of molecular variance (AMOVA) revealed that the variation of N.bulbuliferum among the populations was 77.12% (P<0.01),and the variation within the population was 22.88% (P<0.01).These findings clearly evidenced the genetic relationship of Notholirion in the Hengduan Mountains.Importantly,the high diversity of N.bulbuliferum found in this study indicated that reasonable measures should be taken for the conservation and restoration for medicinal and ornamental plant species.